Methods of reducing beta-amyloid polypeptides

ABSTRACT

The invention features methods of reducing the level of a β-amyloid (Aβ) polypeptide using an Cimicifuga extract. The invention also features methods of producing an active fraction from an extract of Cimicifuga. The invention further features a composition containing an active fraction of a Cimicifuga extract and an article of manufacture containing such a composition.

TECHNICAL FIELD

[0001] This invention relates to β-amyloid (Aβ) polypeptides, and moreparticularly to methods of reducing Aβ polypeptides using an extract, oractive fraction thereof, of Cimicifuga.

BACKGROUND

[0002] Patients with Alzheimer's disease (AD) are typically presented toa clinician by a relative who has observed a decline in memory with orwithout a change in other cognitive abilities such as declines inexecutive function, difficulty in word finding or visuo-spatialimpairment such as an inability to draw complex geometric structures orbecoming lost in familiar places. Neuropathological analysis of brainsfrom patients with AD has revealed extensive neuronal and synaptic lossin select brain regions, neurofibrillary tangles, and the deposition ofβ-amyloid (Aβ) polypeptides in the form of senile plaques throughout thehippocampus and neocortex.

[0003] Aβ polypeptides are produced from the amyloid precursor protein(APP) through the combined proteolytic actions of β- and γ-secretasesand are then secreted into the extracellular milieu. Biochemical andimmunocytochemical studies have revealed that the Aβ polypeptidesdeposited in brains from patients with AD have substantial amino- andcarboxyl-terminal heterogeneity and can contain from 39 to 43 amino acidresidues, with β-amyloid 1-40 (Aβ40) and 1-42 (Aβ42) being the mostpredominant. By comparing signals obtained using antibodies directed atthe amino terminus of Aβ with those obtained by capturing the peptidefrom an internal epitope, it was found that virtually all Aβ42 in ADbrain is amino-terminally modified and/or truncated.

SUMMARY

[0004] The invention features methods of reducing the level of an Aβpolypeptide using a Cimicifuga extract. The invention also featuresmethods of producing an active fraction from an extract of Cimicifuga.An active fraction is one that is able to reduce the level of an Aβpolypeptide in or secreted from a cell (in vitro or in vivo). Theinvention further features a composition containing an active fractionof a Cimicifuga extract and an article of manufacture containing such acomposition.

[0005] In one aspect, the invention provides methods of reducing thelevel of a β-amyloid (Aβ) polypeptide in or secreted from a cell. Themethods include contacting the cell with an amount of a Cimicifugaextract or of an active fraction thereof in an amount that is effectivefor reducing the level of the Aβ polypeptide and monitoring the level ofthe Aβ polypeptide in or secreted from the cell. Representative cellsinclude H4 cells, M17 cells, 293 cells, Chinese hamster ovary (CHO)cells, primary fibroblasts, C6, primary neuronal, primary mixed braincultures, Daoy, SK-N-SH, SK-N-AS and SK-N-FI.

[0006] In another aspect, the invention provides methods for reducingthe level of an Aβ polypeptide in a mammal, including administering anamount of a Cimicifuga extract or an active fraction thereof to themammal in an amount that is effective for reducing the level of the Aβpolypeptide and monitoring the level of the Aβ polypeptide in themammal.

[0007] In another aspect of the invention, there are provided methods oftreating a mammal having AD or at risk to develop AD. The methodsinclude administering an amount of a Cimicifuga extract or an activefraction thereof to the mammal in an amount that is effective fortreating or preventing AD. Generally, the extract or active fractionthereof is administered to a mammal orally, intravenously,intracranially, intracerebrally, subcutaneously, intramuscularly,intranasally or intraperitoneally. A representative mammal is a rodent,for example, a mouse. In one embodiment, the mouse expresses an APPcarrying a Swedish mutation. An example of such a mouse is a Tg2576mouse.

[0008] The Cimicifuga extract or active fraction thereof can be from C.racemosa, and further can be obtained from the root or rhizome of a C.racemosa plant. The C. racemosa extract is generally an ethanolic or anaqueous extract. An active component within an active fraction can besoluble in a solvent such as methylene dichloride, ethyl acetate andn-butanol, and active components soluble in such solvents are typicallylipophilic. An active component within an active fraction that iscapable of reducing the level of an Aβ polypeptide can have a molecularweight of less than 10 kD.

[0009] A reduction in the level of an Aβ polypeptide can be due todecreased production of the Aβ polypeptide or increased catabolism ofthe Aβ polypeptide. In an embodiment of the invention, the level of theAβ polypeptide is reduced by at least 10%, at least 25%, at least 50%,or at least 80% compared to the level of the Aβ polypeptide in orsecreted from a corresponding cell not contacted with the extract oractive fraction thereof. A reduction in the level of an Aβ polypeptidecan be a reduction in the level of Aβ40 or Aβ42. Further, the reductionin the level of an Aβ polypeptide can be a preferential reduction in thelevel of Aβ42. In addition, the extract or active fraction thereoftypically should have no significant effect on the level of one or moreof APP, CTFα, CTFβ, or sAPPα.

[0010] In yet another aspect of the invention, there are providedmethods of producing an active fraction of a Cimicifuga extract thatreduces the level of an Aβ polypeptide upon contact with a cell. Themethods include obtaining an extract of Cimicifuga plant material,size-fractionating the extract through a filter to obtain an activefraction, and testing the active fraction to confirm that the activefraction reduces the level of an Aβ polypeptide. Generally, an activefraction contains active components having a molecular weight of lessthan about 10 kD.

[0011] The invention also provides methods of producing an activefraction of a Cimicifuga extract that reduces the level of an Aβpolypeptide upon contact with a cell, including obtaining an extract ofCimicifuga plant material, extracting the Cimicifuga extract with hexane(thereby producing a hexane-soluble fraction and a hexane-insolublefraction) and testing the hexane-insoluble fraction to confirm that thehexane-insoluble fraction reduces the level of the Aβ polypeptide. Arepresentative hexane is n-hexane. Such methods can additionally includeextracting the hexane-insoluble fraction with a dichloroalkane(producing a dichloroalkane-soluble fraction and adichloroalkane-insoluble fraction) where the dichloroalkane-solublefraction typically reduces the level of the Aβ polypeptide upon contactwith a cell. A representative dichloroalkane is methylene dichloride.Methods of producing an active fraction also can include extracting thedichloroalkane-soluble fraction with an alkylacetate (producing analkylacetate-soluble fraction and an alkylacetate-insoluble fraction)where the alkylacetate-soluble fraction typically reduces the level ofan Aβ polypeptide. A representative alkylacetate is ethyl acetate.Further, methods of the invention can include extracting thealkylacetate-soluble fraction with an alcohol (producing analcohol-soluble fraction and an alcohol-insoluble fraction) wherein thealcohol-soluble fraction typically reduces the level of the Aβpolypeptide. A representative alcohol is n-butanol. The product of anyof the above-described extractions or fractionations can be furtherconcentrated, for example, by lyophilizing.

[0012] In another aspect of the invention, there is provided acomposition containing an active fraction of a Cimicifuga extract and apharmaceutically acceptable carrier. Such an active fraction can reducethe level of an Aβ polypeptide upon contact with a cell producing the Aβpolypeptide.

[0013] In still yet another aspect of the invention, there is providedan article of manufacture containing an active fraction of a Cimicifugaextract, a pharmaceutically acceptable carrier, and packaging material.Generally, packaging material contains a label or package insertindicating that the composition is effective for reducing the level ofan Aβ polypeptide.

[0014] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

[0015] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe drawings and detailed description, and from the claims.

DETAILED DESCRIPTION

[0016] The invention provides methods of reducing the level of an Aβpolypeptide in or secreted from a cell. Methods of the invention includecontacting a cell with an extract of Cimicifuga or an active fractionthereof in an effective amount such that Aβ is reduced, and subsequentlymonitoring the level of Aβ.

[0017] Traditional Chinese medicine has long employed Cimicifuga speciesfor the treatment of menstrual cramps, fatigue, anxiety, rheumatoidarthritis, alleviation of fever, pain, inflammation, sedation, swellingof joints, respiratory congestion from colds, and high blood pressure.More than two centuries ago, Native Americans were using the root ofCimicifuga racemosa, also known as black cohosh, black snakeroot,bugbane, bugwort, and squawroot, to relieve many of the symptomsassociated with menstruation and menopause, including cramps, hotflashes, headaches, irritability, sweats, as well as many of thesymptoms unrelated to menstruation discussed above. Cimicifuga, aperennial native woodland plant, includes several commonly known speciesthat are used medicinally and/or ornamentally (e.g., C. simplex, C.dahurica, C. foetida, C. japonica, C. acerina, and C. racemosa).Extracts or active fractions of C. racemosa are particularly useful inthe methods of the invention.

[0018] Cimicifuga Extracts and Reduction of Aβ Polypeptide Levels

[0019] The invention features methods of reducing the level of an Aβpolypeptide in or secreted from a cell. As used herein, an Aβpolypeptide refers to a portion of an APP (e.g., human APP) that isproduced following cleavage by β-secretase and γ-secretase (for example,residues 671-711 (Aβ40) or 671-713 (Aβ42) of GenBank Accession No.D87675). An Aβ polypeptide can have from 39 to 43 amino acid residues(e.g., 39, 40, 41, 42 or 43 residues). Given the heterogeneity in thelength of Aβ polypeptides, it is a further feature of the invention thatan extract can reduce the level of a specific Aβ polypeptide (e.g., Aβ40or Aβ42). The ability to distinguish a reduction in the level of, forexample, Aβ40 or Aβ42 is significant, since Aβ42 is considered to bemore amyloidogenic. For example, immunocytochemical analysis has shownimmunolabeling for Aβ42 in all types of senile plaques, poor labeling ofplaques using Aβ40 end-specific antibodies, and detection of both theAβ40 and Aβ42 epitopes in cerebrovascular amyloid. Moreover, analysis offamilial AD (FAD)-linked mutations has shown elevations in extracellularAβ accumulation, particularly Aβ42. As described herein, the reductionof Aβ polypeptides by Cimicifuga extracts can be preferential for Aβ42.As used herein “preferential” refers to a significant reduction of oneAβ polypeptide (e.g., Aβ42) compared to the level of one or moredifferent Aβ polypeptides (e.g., Aβ40). “Significant” refers to astatistical calculation of significance in which a p-value of less than0.05 (e.g., a p-value of less than 0.025 or less than 0.01) is obtainedusing an appropriate statistical calculation, e.g., a paired t-test.Therefore, a p-value of greater than 0.05 indicates a lack ofstatistical significance based on such a calculation.

[0020] Methods of the invention include contacting a cell (in vitro orin vivo) with an effective amount of a Cimicifuga extract or activefraction thereof and monitoring the level of Aβ in or secreted from thecell. An effective amount of an extract or an active fraction is anamount that reduces the level of an Aβ polypeptide in or secreted from acell by at least 10% (e.g., at least 20%, 25%, 30%, 40%, 50%, 60%, 70%,80%, or 90%) or completely abolishes the level of an Aβ polypeptidecompared to the level of the Aβ polypeptide in or secreted from acorresponding cell not treated with the extract or active fraction. Acomponent in an active fraction that reduces the level of an Aβpolypeptide can exert an effect at any of a number of steps along thepathway of Aβ secretion and, in the case of AD, deposition in the brain.The level of Aβ polypeptide not only depends upon its production, butalso on the mechanisms responsible for its removal. Without being boundby a particular mechanism, a reduction in the level of an Aβ polypeptidecan be due to the activity of binding proteins that sequester thepolypeptide, or to other cellular mechanisms such as decreasedproduction or increased catabolism of an Aβ polypeptide. By way ofexample, catabolism of an Aβ polypeptide likely involves bothintracellular (e.g., acting at the site of Aβ polypeptide generationand/or within the secretory pathway) and extracellular (e.g.,cell-surface, secreted, endosomal and/or lysosomal) proteases. Compoundsthat reduce the level of one or more Aβ polypeptides can be useful, forexample, as therapeutic compounds or to design therapeutic compounds.

[0021] Cells can be contacted in vitro with a Cimicifuga extract oractive fraction thereof while in cell culture (e.g., introducing anextract into culture media). Representative cells that can be usedinclude, but are not limited to, H4 neuroglioma, M17 neuroblastoma,human kidney 293, Chinese hamster ovary (CHO), primary fibroblast, C6,primary neuronal, primary mixed brain, Daoy, SK-N-SH, SK-N-AS andSK-N-FI-cells. Cells suitable for primary screening can be obtained fromthe American Type Culture Collection (ATCC) (10801 University Blvd.,Manassas, Va.20110). In addition, such cells can be transgenic cellscarrying a construct containing a nucleic acid encoding APP or anyportion of APP containing an Aβ fragment. For example, H4βAPP695 wtcells are H4 cells carrying a human wild-type 695-amino acid APPisoform. Isoforms of APP that are produced by alternate mRNA splicinginclude the aforementioned APP695 as well as APP751 and APP770. Inaddition, cells can express human APP carrying a mutation that, forexample, changes a lysine-methionine at amino acid residues 670 and 671to an asparagine-leucine (i.e., the Swedish mutation) or one thatchanges a valine to an isoleucine at amino acid residue 717.

[0022] A Cimicifuga extract or active fraction therefrom also can beadministered to a mammal such as a human or a rodent to reduce the levelof Aβ. For example, a Cimicifuga extract can be administered to anindividual over the age of 60, or to an individual diagnosed with AD orthat is at risk for developing AD (e.g., based upon familial history orthe presence of a mutation associated with AD). A Cimicifuga extractalso can be administered to a rodent such as a guinea pig or a mouse,and in particular, to an animal model of AD. Animal models of AD areavailable that accumulate Aβ and that develop plaques in anage-dependent manner. In particular, transgenic mice are available thatcarry a mutation in a presenilin gene and/or express an APP carrying amutation (for example,,a Swedish mutation). A representative mousecarrying a Swedish mutation is Tg2576 (Holcomb et al., 1998, Nat. Med.,4:97-100). The Tg2576 mouse accumulates Aβ polypeptides and developsplaques in an age-dependent manner. A Cimicifuga extract or activefraction thereof can be administered to a mammal by any route, includingorally, intravenously, intracranially, intracerebrally, subcutaneously,intramuscularly, intranasally or intraperitoneally.

[0023] Since a reduction in the level of an Aβ polypeptide in vitro orin vivo can be a direct result of a decrease in cell viability, toxiceffects of a Cimicifuga extract or active fraction thereof on a cell ora mammal are typically evaluated. Methods to evaluate cellular toxicityare known to those of skill in the art. For example, the degree ofconversion of a tetrazolium salt (e.g., MTS) into formazan is directlyproportional to the number of metabolically active cells on a cellculture plate. Conversion of MTS can be measured using the CeIlTiter 96®assay kit (Promega; Madison, Wis.). Further, the number of lysed cellsis quantitatively proportional to the LDH (lactatedehydrogenase)-catalyzed conversion of tetrazolium salt to red formazan.The Cytotox 96 assay kit (Promega) can be used to examine LDH release.

[0024] To address issues of toxicity in vivo, standard haematoxylin/eosin (H/E) staining can be performed on the liver, kidney, spleen, andbrain of an animal (e.g., a rodent such as an animal model of AD) tolook for any abnormalities apparent between the extract-treated andcontrol groups. In addition, serum levels of blood urea nitrogen (BUN),aspartate amino transaminase (AST-SGOT), and alkaline phosphatase (ALP)can be monitored in an animal to evaluate renal function, liver damage,and drug-induced biliary obstructions.

[0025] Methods of Detecting Aβ Polypeptides

[0026] Methods for detecting Aβ in cell culture or in a biologicalsample from an individual (e.g., plasma or cerebrospinal fluid) areknown to those of skill in the art. High throughput screens have beendeveloped to examine the level of Aβ40 or Aβ42 in medium conditioned bya cell line over time. A highly specific sandwich ELISA is describedherein using a BAN-50 antibody, which specifically captures Aβ at theN-terminus, and then either a BA-27 antibody, which detects onlyfull-length Aβ polypeptides ending at position 40, or a BC-05 antibody,which detects only full-length Aβ polypeptides ending at position 42(Suzuki et al., 1994, Science, 264:1336-40; Asami-Odaka et al., 1995,Biochem., 34:10272-8).

[0027] Suitable antibodies that detect an epitope within Aβ (or withinthe Aβ portion of APP) are also commercially available from a variety ofsources, including, but not limited to, Biosource International(Camarillo, Calif.), Senetek PLC (London, England), Zymed Laboratories(San Francisco, Calif.), Peninsula Laboratories (San Carlos, Calif.) andBoehringer Mannheim (Indianapolis, Ind.). Several of the commercialsources listed herein also provide antibodies with specific bindingaffinity for either Aβ40 or Aβ42. In addition, BNT-77 is an antibodythat captures rodent Aβ (Asami-Odaka et al., 1995, Biochem., 34:10272-8)that can be used in conjunction with the BA-27 or BC-05 antibodiesdescribed above.

[0028] In addition to the specific sandwich ELISA described herein,other types of solid phase. immunoassays as well as immunoassay formatssuch as Western blots or immunoprecipitations may be used to detect Aβand are well known in the art. See, Short Protocols in MolecularBiology, Ch. 11, John Wiley & Sons, Ed., Ausubel et al., 1992.Solid-phase immunoassays include competition immunoassays,immobilized-antigen immunoassays, immobilized-antibody immunoassays, anddouble-antibody immunoassays. For example, a typical double-antibodyimmunoassay to detect Aβ can include the following steps: attaching anantibody with binding affinity for Aβ to a solid support; exposing theantibody to unlabeled Aβ polypeptide; washing to remove unbound Aβ; andquantitating the amount of Aβ bound to the immobilized antibody using anexcess of a second antibody. The second antibody can have bindingaffinity for Aβ and can be radiolabeled or conjugated to a chemical orenzyme for detection following addition of an appropriate substrate.

[0029] Western blotting to detect Aβ typically includes the steps ofelectrophoretically separating peptides in or secreted from a cell;transferring the peptides from the separation medium (e.g., a gel) to asolid support (e.g., nitrocellulose, nylon); and probing with antibodieswith binding affinity for Aβ. Probing can be direct (e.g., a labeledprimary antibody) or indirect (e.g., an unlabeled antibody specific forAβ, which is subsequently detected with a labeled secondary antibody orimmunological reagent, for example, protein A or anti-immunoglobulin).

[0030] Aβ immunoprecipitation methods generally include the followingsteps: radiolabeling cells expressing Aβ; lysing the cells; formingspecific immune complexes between Aβ and an antibody with bindingaffinity for Aβ; collecting and purifying the immune complexes; andanalyzing the radiolabeled Aβ in the immunoprecipitate.Immunoprecipitation is often used to detect and quantitate targetantigens in complex mixtures of proteins. Immunoprecipitation can beused to analyze unlabeled proteins from unlabeled cells, providedsufficiently sensitive methods are available to detect the targetprotein after it has been dissociated from the antibody.

[0031] A detectable label, e.g., a radioactive label (e.g., ³H, ¹²⁵I,¹³¹I, ³²P, ³⁵S, and ¹⁴C) or a non-radioactive label (e.g., a fluorescentlabel, a chemiluminescent label, a paramagnetic label, or an enzymelabel) may be attached to an antibody or a fragment thereof usingtechniques known to those of ordinary skill in the art. Examples ofenzyme labels used routinely in the art for detection and quantitationinclude horseradish peroxidase (HRP) and alkaline phosphatase (AP). Thesubstrates available for either HRP or AP labels are known in the artand can be selected based upon the desired method of detecting complexformation (e.g., a fluorogenic, chemiluminescent or calorimetricsignal).

[0032] Aβ that has been detected by any of the methods described hereinor other methods known to those of skill in the art can be visualized orquantitated using methods routine in the art, including autoradiographyof a radioactive label (e.g., x-ray film, phosphorimaging, ordensitometric analysis) and spectrophotometry of a fluorescent label orof a colorimetric reaction produced by, for example, an enzymatic label.In addition, a non-Aβ polypeptide also can be detected and quantitatedusing methods similar to those described herein for Aβ (e.g., fornormalization purposes).

[0033] In addition, mass spectrometry (MS) can be used to detect andquantitate Aβ polypeptides. Several types of MS are available androutinely used in the art, and include Fourier-transform MS, Ion-trapMS, Magnetic-sector MS, Quadropole MS and Time-of-flight (TOF) MS. Byway of example, Ciphergen (Fremont, Calif.) sells a biochip system forcapturing Aβ polypeptides from culture medium or a biological sample andutilizes SELDI technology (Surface-Enhanced Laser Desorption/Ionization)with TOF-MS to detect arid quantitate the level of Aβ polypeptides.

[0034] Neuritic plaques in the brain of an individual with AD can bedetected and/or monitored in vivo using an Aβ polypeptide covalentlymodified with a polyamine (e.g., putrescine, spermidine, or spermine)(Wengenack et al., 2000, Nat Biotechnol., 18:868-72). A radiolabeledpolyamine-modified Aβ polypeptide can be administered to an individualintravenously and detected using standard methods. For example, aradiolabel suitable for diagnostic imaging can be used (e.g., ¹²³I) anddetected using single photon emission computed tomography (SPECT).

[0035] Producing Cimicifuga Extracts and Active Fractions Thereof

[0036] Extraction is a process whereby the desired constituents of aplant or plant part are removed using a solvent or other means.Generally, an extract of Cimicifuga is obtained from the root or rhizomeof the plant, although leaves, stems and flowers also can be used. TheCimicifuga extract or active fraction can be from C. racemosa. Toproduce an extract, plant, material is usually first cleaned and driedif necessary. Drying can be done naturally (e.g., by air drying) orartificially (e.g., using warm-air fans or conveyor dryers). The plantmaterial then can be ground, cut, or shredded using, for example, hammeraction, pressure, friction or impact cutting. Methods of removing thedesired constituents from the plant material include, but are notlimited to, organic solvent extraction, supercritical gas extraction,and steam distillation. By way of example, there are a number ofprocedures for organic solvent extraction, including maceration (soakingand agitating the plant material with a solvent), percolation (repeatedrinsing of the plant material with a solvent), and countercurrentextraction (continuous flow of a solvent in the opposite direction asthe plant material). Representative solvents include, but are notlimited to, ethanol, benzene, toluene and ether. Aqueous extracts, suchas decoctions (boiling the plant material, generally used for hardtissues), infusions (steeping the plant material, generally used forsoft tissues) or macerations, can also be produced, although microbialcontamination can be a concern with aqueous extraction methods. As usedherein in the methods of the invention, a C. racemosa extract can be anethanolic extract or an aqueous extract, depending upon the solubilityof an active component that reduces the level of an Aβ polypeptide.Extracts of Cimicifuga are commercially available, and includeRemifemin® (SmithKline Beecham, Research Triangle Park, N.C.), and BlackCohosh (Viable Herbal Solutions, Morrisville, Pa.; or Whole HealthDiscount Center, http://www.WholeHealthDiscountCenter.com).

[0037] Methods of producing active fractions (i.e., containing one ormore active components) from a Cimicifuga extract are provided by theinvention. Active components of a Cimicifuga extract or active fractioncan include, but are not limited to, polyphenols, flavonoids, aromaticacids, metabolites, alkaloids, proteins, carbohydrates, starches,steroids, resins, elements or combinations thereof (e.g., glycoproteins)that, alone or in combination with other components, can reduce thelevel of an Aβ polypeptide. For example, fractionating by traditionalsolvent extraction employs partitioning of a solute between twoimmiscible phases, typically an organic phase (e.g., n-hexane, methylenedichloride, ethyl acetate or n-butanol) and an aqueous phase. Rapidextraction kinetics and the ability to utilize a number of differentdiluents, extractants, and aqueous phases makes solvent extraction apowerful separation method. In addition, numerous other separationprocedures can be employed to further purify desired components orremove unwanted or contaminating components, including decanting,filtration, sedimentation, centrifugation, heating, adsorption,precipitation, chromatography, or ion exchange. The resulting activefraction can be subsequently concentrated by evaporation, vaporization,lyophilization or vacuum drying. Although the invention exemplifiesfractionating using organic solvent partitioning, those of skill in theart are aware of the advantages of using certain separation techniquesin combination with others to increasingly partition one or more activecomponents into active fractions.

[0038] One method of the invention includes obtaining an extract ofCimicifuga plant material and size-fractionating the extract, e.g.,through a filter. The flow-through represents an active fraction. Usingthis or a similar method of fractionating a Cimicifuga extract, anactive component within the active fraction can pass through a 10kilodalton (kD) nominal molecular weight filter or concentrator.

[0039] Another method of producing an active fraction of a Cimicifugaextract includes obtaining an extract of Cimicifuga plant material, andextracting the extract with hexane (e.g., n-hexane). Thehexane-insoluble fraction can be used to reduce the level of an Aβpolypeptide following contact with a cell or can be furtherfractionated. For example, the hexane-insoluble fraction can beextracted with a dichloroalkane (e.g., methylene dichloride ordichloroethane). As described herein, the dichloroalkane-solublefraction is able to reduce levels of Aβ. An active component within thedichloroalkane-soluble fraction can be further partitioned by extractingwith an alkylacetate (e.g., ethyl acetate or methyl acetate) andobtaining an alkylacetate-soluble fraction. An active component withinthe alkylacetate-soluble fraction can be further partitioned byextracting with an alcohol solvent (e.g., n-butanol or 1-pentanol) andobtaining the alcohol-soluble fraction. Those of skill in the art areaware of additional steps during fractionating that can facilitatesample handling (e.g., concentrating an active fraction).

[0040] Given the high throughput cell based assays described herein,hundreds or thousands of fractions or combinations of fractions can berapidly screened to identify those containing one or more activecomponents or to identify fractions that, when combined, conferactivity. An animal such as the Tg2576 mouse or a transgenic mousecarrying a Swedish mutation and a mutation in a presenilin sequence alsocan be used to evaluate the level of Aβ (e.g., in plasma or homogenizedbrain using, for example, the sandwich ELISA described herein), plaqueburden (e.g., in brain tissue using, for example, immunocytochemicalanalysis), behavior/memory (e.g., using a Morris water maze to evaluatespatial reference memory), or other neurological markers such asdifferences in astroglia, dystrophic neuritis, microglia and the stateof tau phosphorylation (e.g., by immunocytochemical analysis of braintissue) following administration of a Cimicifuga extract or activefraction.

[0041] An active component from a Cimicifuga extract or active fractionthat is able to reduce Aβ levels can be soluble in methylene dichloride,ethyl acetate and n-butanol, generally indicating a lipophilic activecomponent. Lipophilic compounds are those that prefer a nonpolarenvironment to an aqueous one. Compounds from C. racemosa extract havebeen identified previously using methods such as those described herein,and include a phytoestrogen capable of binding estrogen receptors, andcimicifugin and macrotin resins, which effect, among others, thereproductive and nervous systems. Also identified from Cimicifugaextracts are triterpene glycosides, including cimicifugoside, which isbelieved to affect the hypothalamus-pituitary system and thereproductive and nervous systems, actein, a steroidal derivative thatlowers blood pressure in animals, 27-deoxyactein, and racemoside. Anisoflavone called formonenetin has been identified as binding toestrogen receptors in the rat uterus. Aromatic acids, including ferulicacid and isoferulic acid are believed responsible for the extract'santi-inflammatory effects.

Compositions and Articles of Manufacture

[0042] The invention further features a composition containing an activefraction of a Cimicifuga extract. A composition containing an activefraction of a Cimicifuga extract can be in any form provided thecomposition can be placed in contact with a cell in an amount and for alength of time effective to reduce the level of an Aβ polypeptide.

[0043] Compositions of the invention may be administered on a continuousor an intermittent basis. By way of example, a composition within thescope of the invention can be in the form of a liquid, solution,suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream,nebulae, mist, atomized vapor, or aerosol. For the purpose of thisinvention, routes of administration include, but are not limited to,oral, nasal, intravenous, intramuscular, intraperitoneal, subcutaneous,intrathecal, intradermal, or topical. The route of administration candepend on a variety of factors, such as the environment in which cellsare contacted and therapeutic goals. The dosages of a particularcomposition will depend on many factors, including the mode ofadministration and the cells being treated. Typically, the amount of anactive fraction contained within a single dose of a composition will bean amount that effectively reduces the level of an Aβ polypeptidewithout inducing significant toxicity.

[0044] In addition, compositions within the scope of the invention cancontain a pharmaceutically acceptable carrier for in vivo administrationto a mammal, including, without limitation, sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and injectable organic esters.Aqueous carriers include water, alcohol, saline, and buffered solutions.Pharmaceutically acceptable carriers can also include physiologicallyacceptable aqueous vehicles (e.g., physiological saline or artificialcerebral-spinal fluid) or other known carriers appropriate to specificroutes of administration. Additional compounds can be included in acomposition, such as steroids, mucolytic agents, anti-inflammatoryagents, immunosuppressants, dilators, vasoconstrictors, or combinationsthereof. Preservatives, flavorings, and other additives such as, forexample, anti-microbials, anti-oxidants, chelating agents, inert gases,and the like may also be present in a composition.

[0045] For oral administration, tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents, fillers, lubricants, disintegrants, or wetting agents.The tablets can be coated by methods known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspension, or can be presented as a dry productfor constitution with saline or other suitable liquid vehicle beforeuse. Liquid preparations also can contain pharmaceutically acceptableadditives such as suspending agents, emulsifying agents, non-aqueousvehicles, preservatives, buffer salts, flavoring, coloring andsweetening agents as appropriate. Preparations for oral administrationcan be suitably formulated to give controlled release of the compound.

[0046] The invention further provides for an article of manufacture thatincludes a composition containing an active fraction of a. Cimicifugaextract, and packaging material. The packaging material in or on anarticle of manufacture indicates that the composition therein iseffective for reducing the level of an Aβ polypeptide. Components andmethods for producing articles of manufactures are well known in theart. Instructions, for example on a label or package insert, describinga particular dose of the composition to be administered may be includedin such kits. Different kits may be manufactured that contain acomposition in different forms (e.g., a pill or a liquid) or thatcontain a dose of a composition appropriate to reduce the level of an Aβpolypeptide in, for example, an adult female subject or in an adult malesubject (based upon an average weight). Instructions can further includea table or chart for adjusting a particular dose of a composition for asubject that deviates from average.

[0047] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Primary Screens to Identify Cimicifuga Extracts orActive Fractions Thereof that Reduce the Level of an Aβ Polypeptide invitro

[0048] Primary screens with cell cultures used a Cimicifuga ethanolicextract (obtained from Nature's Answer (www.naturesanswer.com) orNature's Apothecary (Louisville, Colo.)). Nature's Answer providedethanolic Cimicifuga extracts at 10-14% or 15-20%, while the Cimicifugaextract provided by Nature's Apothecary was at 55% ethanol. Anappropriate dilution of each extract at a final concentration of 0.005%,0.05% or 0.5% was placed into culture medium in a 96-well low bindingmicrotiter library plate at the appropriate dilution. Vehicle controls,and 30 mM acetamidophenol were included in separate wells on eachlibrary plate to determine any vehicle-induced alterations in the levelof Aβ, and for toxicity assessment, respectively (acetaminophen has beenshown to be toxic to cells). The diluted Cimicifuga extracts andcontrols from the library plate were then directly placed onto confluentH4βAPP695 wt cells in a 96-well cell culture plate by multi-channelpipette. Following incubation for 12 hrs, the medium was removed bymulti-channel pipette and aliquots immediately placed directly on96-well sandwich ELISA plates for the measurement of Aβ40 or Aβ42 and ona 96-well LDH assay plate. The cells were then washed and subjected to a96-well MTS assay.

[0049] In particular, the sandwich ELISA used herein was performed asfollows: 96-well microtiter plates were coated overnight at 4° C. with100 μl of a 5 μg/ml dilution of primary antibody in sodium carbonatecoating buffer (SCCB; 0.1 M Na₂CO₃, pH 9.6). Plates were blockedovernight at 4° C. with 300 μl of Block Ace Solution (PBS+1.0% Block Ace(Snow Brand Milk Products, Japan), 0.05% NaN₃, pH 7.4). Samples foranalysis and synthetic Aβ standards (Bachem, Switzerland) were dilutedin buffer EC (0.02 M NaH₂PO₄, 0.002 M EDTA, 0.4 M NaCl, 0.2% BSA, 0.05%CHAPS, 0.04% Block Ace, 0.05% NaN₃, pH 7.0) and allowed to incubate onthe plates overnight at 4° C. Plates were washed twice with PBS (8 mMNa₂HPO₄, 1.5 mM KH₂PO₄, 139 mM NaCl, 2.7 mM KCl, pH 7.4) and 100 μl ofsecondary antibody directly coupled to HRP (Pierce EZ-link PlusActivated Peroxidase kit, according to manufacturers directions; PierceChemical Co., Rockford, Ill.) was allowed to bind either 4 hrs at roomtemperature or overnight at 4° C. Plates were then washed twice with PBScontaining 0.05% Tween 20 followed by two additional washes in PBS.Detection was performed using the TMB (3,3′,5,5′-tetramethyl-benzidine)as an HRP substrate according to the manufacturer's specifications(Kirkegaard & Perry Laboratories (KPL), Gaithersburg, Md.) and thereaction stopped by the addition of 100 μl of 1N H₃PO₄. Plates were readat 450 nm in a SpectraMax Plus spectrophotometer (Molecular Devices;Sunnyvale, Calif.) and analyzed by SOFTmax® PRO software. Aβ40 or Aβ42were quantitated by comparison with the values obtained for eachsynthetic Aβ standard from the same plate.

Example 2 Dose Response Analysis of C. racemosa Extract on the Level ofAβ40 and Aβ42 Polypeptides

[0050] A statistically significant reduction in the level of Aβ42 wasobserved at all concentrations and with either C. racemosa extractexamined. The specific effect on Aβ42 plateaued at approximately 50%inhibition. At concentrations greater than 0.15%, the level of Aβ40 alsobegan to decrease in the presence of C. racemosa extract. The reductionin Aβ40 observed at the higher doses might be due to the presence of oneor more other components within the extract that reduce either total Aβor specifically Aβ40, or to overlapping effects on Aβ40 and Aβ42 by thesame component.

Example 3 C. racemosa Extract does not Significantly Affect theaccumulation of APP, Carboxyl Terminal Fragment {acute over (α)} (CTFα)CTFβ, or sAPPα

[0051] APP, CTFβ and CTFα were analyzed in detergent-extracted celllysates by Western blotting using an antibody directed against the last20 amino acids of APP (provided by Dr. K. Sambamurti, Mayo Clinic,Jacksonville, Fla.). CTFβ is produced, along with sAPPβ, followingβ-secretase cleavage of APP, while CTFα and sAPPα are produced followingcleavage of APP by α-secretase. sAPPα was detected in conditioned mediumusing the BAN-50 antibody against Aβ residues 1-16. The C. racemosaextract did not have a significant effect on the steady state levels ofAPP, CTFα, CTFβ, or sAPPα. Therefore, the effect of C. racemosa extracton Aβ42 was specific with respect to effects on APP processing.

Example 4 Multiple lots of C. racemosa Extract from Different VendorsShow Specific Reductions in Aβ42

[0052] To examine the uniformity of C. racemosa extract preparations,the variance between manufacturers' extracts was examined. H4βAPP695 wtcells were treated with C. racemosa ethanolic extracts obtained from 2separate manufacturers (Nature's Answer, 10-14% or 15-20% ethanol; orNature's Apothecary, 55% ethanol). Cells were treated with a finalconcentration of 0.1% of C. racemosa extract. C. racemosa extracts fromeach manufacturer showed a similar reduction in Aβ42.

Example 5 Secondary Screens Using Tg2576 Mice to Identify Extracts orActive Fractions thereof that Reduce the Level of an Aβ Polypeptides invivo

[0053] First, a dealcoholized extract or active fractions thereof areadministered to young, aged matched, 4 week-old female Tg2576 mice. Theeffect of administration of an appropriate vehicle is used for controlanimals. Following a single administration of the test extract bygavage, the mice are sacrificed at defined time intervals and the brainsanalyzed for Aβ. 4 week-old mice are chosen so that Aβ is analyzed inthe absence of any detectable deposition. 24 mice are assigned to aworking dose group and the same number to a maximal dose group. For theworking dose group, the dose of the extract given to the mice is equalto the body weight adjusted maximum dose reported for use in humans. Forthe maximal dose group, 250 μl is administered (i.e., the maximum volumethat can be safely administered by gavage to mice of this age). 6 miceare sacrificed at each of 6, 12, 24 and 48 hrs post-administration.Plasma is obtained by conventional means from a terminal bleed and thebrain is extracted and flash-frozen for analysis using the sandwichELISA described herein. A significant reduction in Aβ42 is observed inthe treated group when compared to the controls (p<0.05, Mann-Whitney).

[0054] Second, the extract or active fractions are administered for 2wks to 4 week-old mice using the working dose and maximal dose asdescribed above. 6 mice are treated per dose. As drug concentrationapproximates steady state in 4 half lives, this time course allows forsteady state analysis of active components with half lives up to 3-4days.

[0055] Third, mice are started dosing at 4 mo of age (prior todetectable deposition of Aβ) and are dosed for 10 mo until they reach 14mo of age. This age is selected for sacrifice since it is one of theearliest periods where pathology is apparent and behavioral differenceshave been reported. 15 mice are assigned to each testing group. Controlsanimals receive vehicle alone. Mice are evaluated during the 10 moperiod for behavioral changes and at the conclusion of the experiment,they are sacrificed to determine the extent of plaque formation, Aβaccumulation, and other neuropathology apparent in the Tg2576 mouse. Onehalf of the brain is processed for Aβ quantitation by ELISA and one halfis prepared for immunohistochemical analysis. C. racemosa extracttreatment results in a significant reduction in Aβ42 in the older groupof animals (p<0.05, Mann-Whitney).

[0056] The presence of abundant senile plaques throughout thehippocampus and neocortex is a pathological hallmark of Alzheimer'sdisease. A blinded immunohistochemical analysis is performed on both thecontrol and drug-treated mice from the latter group to determine theeffect of C. racemosa extract on senileplaque accumulation. C. racemosaextract treatment results in a significant reduction in the number ofplaques as assessed using antibodies specific to Aβ. In adjacentsections, the majority of the immunoreactive plaques are also positiveby thioflavin-S fluorescent microscopy. The total area occupied by theseplaques is determined by image analysis of immunostained sections. Areduction in the area is observed in the drug-treated mice.

Example 6 Active Fractions of a C. racemosa Extract

[0057] To assess the solubility and determine the molecular size of theactive component in C. racemosa extract, a centrifugation and sizeexclusion experiment was performed. The activity of an ethanolic C.racemosa extract, soluble material following a 15,000 g spin, and theflow-through from a centrifugation using a filtration unit with amolecular weight cut-off of 10 kD were examined in cell cultures. Theseresults indicated that the active component in C. racemosa extract wassoluble and could pass through a 10 kD cut-off filter.

[0058] To examine the stability of the active component in C. racemosaextract, the extract was boiled for 5 min and then analyzed using thecell-based assay in a side-by-side comparison with non-boiled extract.Analysis of the boiled vs. non-boiled extract on H4βAPP695 cells showedan essentially equivalent reduction in the level of Aβ, demonstratingthat an active component within a Cimicifuga extract is heat stabile.

Example 7 Bioassay-guided Identification and Isolation of an ActiveComponent within C. Racemosa Extract

[0059] An ethanolic C. racemosa extract was spun at 15,000 g to removeparticulate material. The supernatant was lyophilized and re-suspendedin distilled water. This suspension was then sequentially extracted inhexane, methylene dichloride, ethylacetate and n-butanol. The solventextracts and the remaining water were aliquoted androtovapped/lyophilized. An aliquot of each was resuspended in ethanol,diluted at several concentrations into H4 cell culture medium andanalyzed for activity in the cell-based assays. Pure ethanol was dilutedsimilarly as a control and the original extract diluted and analyzed inthe cell-based assay to determine any substantial increase in activity.The methylene dichloride-soluble fraction, the ethyl acetate-solublefraction, and the n-butanol-soluble fraction all demonstrated theability to reduce the level of Aβ in the cell-based assay.

[0060] The solvent extract containing activity is then subject topreparative TLC followed by HPLC or HPLC directly as appropriate. HPLCfractions are then analyzed in the cell-based assay. The fraction withthe highest activity is subjected to mass spectral, IR, and NMR analysisfor structure identification. Additional separation steps as discussedherein can be included as necessary.

Other Embodiments

[0061] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of reducing the level of a β-amyloid(Aβ) polypeptide in or secreted from a cell, said method comprisingcontacting said cell with an amount of a Cimicifuga extract or of anactive fraction thereof effective for reducing the level of said Aβpolypeptide in or secreted from said cell and monitoring the level ofsaid Aβ polypeptide in or secreted from said cell.
 2. The method ofclaim 1, wherein said Cimicifuga extract or active fraction thereof isfrom C. racemosa.
 3. The method of claim 2, wherein said C. racemosaextract or active fraction thereof is obtained from the root or rhizomeof a C. racemosa plant.
 4. The method of claim 2, wherein said C.racemosa extract is an ethanolic extract or an aqueous extract.
 5. Themethod of claim 2, wherein an active component within said activefraction is soluble in a solvent selected from the group consisting ofmethylene dichloride, ethyl acetate and n-butanol.
 6. The method ofclaim 5, wherein said active component is lipophilic.
 7. The method ofclaim 2, wherein an active component within said active fraction has amolecular weight of less than 10 kD.
 8. The method of claim 1, whereinsaid cell is selected from the group consisting of H4 cells, M17 cells,293 cells, Chinese hamster ovary (CHO) cells, primary fibroblasts, C6,primary neuronal, primary mixed brain cultures, Daoy, SK-N-SH, SK-N-ASand SK-NFI.
 9. The method of claim 1, wherein said reduction in thelevel of said Aβ polypeptide is due to decreased production of said Aβpolypeptide or increased catabolism of said Aβ polypeptide.
 10. Themethod of claim 1, wherein said level of said Aβ polypeptide is reducedby at least 10% compared to the level of said Aβ polypeptide in orsecreted from a corresponding cell not contacted with said extract oractive fraction thereof.
 11. The method of claim 1, wherein said levelof said Aβ polypeptide is reduced by at least 25% compared to the levelof said Aβ polypeptide in or secreted from a corresponding cell notcontacted with said extract or active fraction thereof.
 12. The methodof claim 1, wherein said level of said Aβ polypeptide is reduced by atleast 50% compared to the level of said Aβ polypeptide in or secretedfrom a corresponding cell not contacted with said extract or activefraction thereof.
 13. The method of claim 1, wherein said level of saidAβ polypeptide is reduced by at least 80% compared to the level of saidAβ polypeptide in or secreted from a corresponding cell not contactedwith said extract or active fraction thereof.
 14. The method of claim 1,wherein said Aβ polypeptide is Aβ40.
 15. The method of claim 1, whereinsaid Aβ polypeptide is Aβ42.
 16. The method of claim 1, wherein saidreduction is preferential for Aβ42.
 17. The method of claim 1, whereinsaid extract or active fraction thereof has no significant effect on thelevel of one or more of APP, CTFα, CTFβ, or sAPPα.
 18. A method forreducing the level of an Aβ polypeptide in a mammal, said methodcomprising administering an amount of a Cimicifuga extract or an activefraction thereof to said mammal effective for reducing the level of saidAβ polypeptide and monitoring the level of said Aβ polypeptide in saidmammal.
 19. The method of claim 18, wherein said extract or activefraction thereof is administered to said mammal orally, intravenously,intracranially, intracerebrally, subcutaneously, intramuscularly,intranasally or intraperitoneally.
 20. The method of claim 18, whereinsaid mammal is a rodent.
 21. The method of claim 20, wherein said rodentis a mouse.
 22. The method of claim 21, wherein said mouse expresses anAPP carrying a Swedish mutation.
 23. The method of claim 22, whereinsaid mouse is a Tg2576 mouse.
 24. A method of treating a mammal havingAD or at risk to develop AD, said method comprising administering anamount of a Cimicifuga extract or an active fraction thereof to saidmammal effective for treating or preventing AD in said mammal.
 25. Amethod of producing an active fraction of a Cimicifuga extract, whereinsaid active fraction reduces the level of an Aβ polypeptide upon contactwith a cell producing said Aβ polypeptide, said method comprising:obtaining an extract of Cimicifuga plant material; size-fractionatingsaid extract through a filter to obtain an active fraction, wherein saidactive fraction comprises active components having a molecular weight ofless than about 10 kD; and testing said active fraction to confirm thatsaid active fraction reduces the level of said Aβ polypeptide uponcontact with said cell producing said Aβ polypeptide.
 26. A method ofproducing an active fraction of a Cimicifuga extract, wherein saidactive fraction reduces the level of an Aβ polypeptide upon contact witha cell producing said Aβ polypeptide, said method comprising: obtainingan extract of Cimicifuga plant material; extracting said Cimicifugaextract with hexane, thereby producing a hexane-soluble fraction and ahexane-insoluble fraction; and testing said hexane-insoluble fraction toconfirm that said hexane-insoluble fraction reduces the level of said Aβpolypeptide upon contact with said cell producing said Aβ polypeptide.27. The method of claim 26, wherein said hexane is n-hexane.
 28. Themethod of claim 26, further comprising: extracting said hexane-insolublefraction with a dichloroalkane, thereby producing adichloroalkane-soluble fraction and a dichloroalkane-insoluble fraction,wherein said dichloroalkane-soluble fraction reduces the level of saidAβ polypeptide upon contact with said cell producing said Aβpolypeptide.
 29. The method of claim 28, wherein said dichloroalkane ismethylene dichloride.
 30. The method of claim 28, further comprising:extracting said dichloroalkane-soluble fraction with an alkylacetate,thereby producing an alkylacetate-soluble fraction and analkylacetate-insoluble fraction, wherein said alkylacetate-solublefraction reduces the level of said Aβ polypeptide upon contact with saidcell producing said Aβ polypeptide.
 31. The method of claim 30, whereinsaid alkylacetate is ethyl acetate.
 32. The method of claim 30, furthercomprising: extracting said alkylacetate-soluble fraction with analcohol, thereby producing an alcohol-soluble fraction and analcohol-insoluble fraction, wherein said alcohol-soluble fractionreduces the level of said Aβ polypeptide upon contact with said cellproducing said Aβ polypeptide.
 33. The method of claim 32, wherein saidalcohol is n-butanol.
 34. The method of claim 26, further comprisingconcentrating said Cimicifuga extract prior to said extraction withhexane, wherein said concentrating comprises lyophilizing saidCimicifuga extract.
 35. A composition, said composition comprising: anactive fraction of a Cimicifuga extract and a pharmaceuticallyacceptable carrier, wherein said active fraction reduces the level of anAβ polypeptide upon contact with a cell producing said Aβ polypeptide.36. An article of manufacture, said article comprising: an activefraction of a Cimicifuga extract, wherein said active fraction reducesthe level of an Aβ polypeptide upon contact with a cell producing saidAβ polypeptide; a pharmaceutically acceptable carrier; and packagingmaterial, wherein said packaging material contains a label or packageinsert indicating that said composition is effective for reducing thelevel of an Aβ polypeptide.